The present invention relates to a cell nucleus observation substrate and a cell nucleus observation apparatus. More particularly, the present invention relates to a cell nucleus observation substrate for fluorescence staining and morphological observing cell nuclei.
In the related art, cell nuclei of cells, microorganisms and the like have been observed using a microscope and the like, and types and properties of the cells and the microorganisms have been determined based on the morphology of the nuclei. For ease of the morphological observation of the nuclei, a pigment for staining the nuclei is used to stain the cells, the microorganisms and the like prior to the observation.
For example, Non-Patent Documents 1 and 2 describe a technology that erythrocytes are stained by Giemsa to observe the cells infected by malaria parasite. In the apparatuses described in Non-Patent Documents 1 and 2, erythrocytes infected by malaria parasite in blood are collected using a magnet, whereby malaria diagnosis can be done conveniently and speedy.
As the pigment for staining the nuclei, methylene blue that is a blue pigment used for the above-mentioned Giemsa stain, hematoxylin that is a blue purple pigment and the like have been used from the past, for example. In recent years, fluorescent pigments bonding to nucleic acids are used to stain the nuclei. Examples of the fluorescent pigments include Hoechst and DAPI.
In relation to the present invention, conventionally known fluorescence as autofluorescence shown by cells upon a fluorescence observation will be described. One of the fluorescence is orange-colored autofluorescence shown by UV-irradiated cells in the presence of copper. For example, it is reported that cells of a particular part of a drosophila larvae midgut emits orange-colored fluorescence when copper is added (see Non-Patent Documents 3 to 11). The cells where the orange-colored fluorescence is especially strongly observed in the drosophila larvae midgut are called as “copper cells” or the like. It is reported that the fluorescence is observed at cells around the copper cells (Non-Patent Document 6) and an entire body wall of the larvae (Non-Patent Document 4) when the concentration of the copper added is increased.
There is a description that the above-mentioned orange-colored fluorescence is observed at both cytoplasms and cell nuclei in cells, and, in particular, is detected predominantly in grains of the cytoplasms (see Non-Patent Documents 4 to 6 and 9). There is a description that a wavelength range of fluorescence is 590 to 630 nm, a peak wavelength is 610 nm and a maximum excitation wavelength is 340 nm (see Non-Patent Document 5).
Also for organisms other than drosophila, autofluorescence having similar properties is observed. For example, there is reported that orange-colored fluorescence (having a peak wavelength of 605 nm) is observed in an individual liver to which copper is added by UV excitation (excitation wavelength of 310 nm) in rat experiments (see Non-Patent Document 11). Furthermore, there is reported that similar fluorescence is observed in a kidney of a model rat having a kidney and a liver where copper is accumulated with aging (see Non-Patent Document 12). Also, the autofluorescence having similar properties is reported in yeast (see Non-Patent Document 13) and human liver tissues of a Wilson's disease patient (see Non-Patent Document 14). The Wilson's disease is a genetic disorder of insufficient excretion of copper and accumulation of copper in liver cells.
As the above-described fluorescent substance emitting orange-colored fluorescence, a composite of copper and metallothionein (MT) (hereinafter abbreviated to as “Cu-MT”) is presumed (see Non-Patent Documents 16 to 25). The Cu-MT has wavelength properties such as an excitation wavelength of 305 nm and a fluorescence wavelength of 565 nm in Non-Patent Document 15, and an excitation wavelength of 310 nm and a fluorescence wavelength of 570 nm in Non-Patent Document 19. It is conceivable that the Cu-MT contain monovalent copper ions (Cu(I)) (see Non-Patent Documents 15, 17, 19, 21 and 25).
As the fluorescent substance containing copper, a compound containing pyrimidine or mercaptide that emits fluorescence by interacting pyrimidine or mercaptide with copper is widely known (see Non-Patent Documents 26 to 31).
On the other hand, an interaction of various metal ions with nucleic acids has been traditionally studied. For example, it is known that when monovalent copper ions are interacted with nucleic acids, a minor amount of copper contained in cell nuclei stabilizes a nucleic acid structure, but hurts DNAs under coexistence of hydrogen peroxide (see Non-Patent Document 32). It is also reported that an interaction with copper changes an absorption spectrum of DNAs (see Non-Patent Documents 32 and 33). Further, it is reported that the change in the absorption spectrum depends on base sequences (specifically, a polymer having a G-C pair and a polymer having an A-T pair) of the DNAs (see Non-Patent Document 32).    Non-Patent Document 1: “Diagnosis of malaria by magnetic deposition microscopy.” Am. J. Trop. Med. Hyg., 2006, Vol. 74, No. 4, p. 568-572    Non-Patent Document 2: “Enhanced detection of gametocytes by magnetic deposition microscopy predicts higher potential for Plasmodium falciparum transmission.” Malaria Journal, 2008, 7, 66    Non-Patent Document 3: Physiological genetic studies on copper metabolism in the genus Drosophila. (1950) Genetics 35, 684-685    Non-Patent Document 4: Organization and function of the inorganic constituents of nuclei. (1952) Exp. Cell Res., Suppl. 2:161-179    Non-Patent Document 5: Ultrastructure of the copper-accumulating region of the Drosophila larval midgut. (1971) Tissue Cell. 3, 77-102    Non-Patent Document 6: Specification of a single cell type by a Drosophila homeoticgene. (1994) Cell. 76, 689-702    Non-Patent Document 7: Two different thresholds of wingless signalling with distinct developmental consequences in the Drosophila midgut. (1995) EMBO J. 14, 5016-5026.    Non-Patent Document 8: Calcium-activated potassium channel gene expression in the midgut of Drosophila. (1997) Comp. Biochem. Physiol. B Biochem. Mol. Biol. 118, 411-420    Non-Patent Document 9: Evidence that a copper-metallothionein complex is responsible for fluorescence in acid-secreting cells of the Drosophila stomach. (2001) Cell Tissue Res. 304, 383-389    Non-Patent Document 10: Peptidergic paracrine and endocrine cells in the midgut of the fruit fly maggot. (2009) Cell Tissue Res. 336, 309-323    Non-Patent Document 11: A luminescence probe for metallothionein in liver tissue:emission intensity measured directly from copper metallothionein induced in ratliver. (1989) FEBS Lett. 257, 283-286    Non-Patent Document 12: Direct visualization of copper-metallothionein in LEC rat kidneys: application of autofluorescence signal of copper-thiolate cluster. (1996) J. Histochem. Cytochem. 44, 865-873    Non-Patent Document 13: Incorporation of copper into the yeast Saccharomyces cerevisiae. Identification of Cu(I)-metallothionein in intact yeast cells. (1997) J. Inorg. Biochem. 66, 231-240    Non-Patent Document 14: Portmann B. Image of the month. Copper-metallothionein autofluorescence. (2009) Hepatology. 50, 1312-1313    Non-Patent Document 15: Luminescence properties of Neurospora copper metallothionein. (1981) FEBS Lett. 127, 201-203    Non-Patent Document 16: Copper transfer between Neurospora copper metallothioneinand type 3 copper apoproteins. (1982) FEBS Lett. 142, 219-222    Non-Patent Document 17: Spectroscopic studies on Neurospora copper metallothionein. (1983) Biochemistry. 22, 2043-2048    Non-Patent Document 18: Metal substitution of Neurospora copper metallothionein. (1984) Biochemistry. 23, 3422-3427    Non-Patent Document 19: (Cu,Zn)-metallothioneins from fetal bovine liver. Chemical and spectroscopic properties. (1985) J. Biol. Chem. 260, 10032-10038    Non-Patent Document 20: Primary structure and spectroscopic studies of Neurospora copper metallothionein. (1986) Environ. Health Perspect. 65, 21-27    Non-Patent Document 21: Characterization of the copper-thiolate cluster in yeast metallothionein and two truncated mutants. (1988) J. Biol. Chem. 263, 6688-6694    Non-Patent Document 22: Luminescence emission from Neurospora copper metallothionein. Time-resolved studies. (1989) Biochem J. 260, 189-193    Non-Patent Document 23: Establishment of the metal-to-cysteine connectivities in silver-substituted yeast metallothionein (1991) J. Am. Chem. Soc. 113, 9354-9358    Non-Patent Document 24: Copper- and silver-substituted yeast metallothioneins: Sequential proton NMR assignments reflecting conformational heterogeneity at the Cterminus. (1993) Biochemistry. 32, 6773-6787    Non-Patent Document 25: Luminescence decay from copper(I) complexes of metallothionein. (1998) Inorg. Chim. Acta. 153, 115-118    Non-Patent Document 26: Solution Luminescence of Metal Complexes. (1970) Appl. Spectrosc. 24, 319-326    Non-Patent Document 27: Fluorescence of Cu, Au and Ag mercaptides. (1971) Photochem. Photobiol. 13, 279-281    Non-Patent Document 28: Luminescence of the copper-carbon monoxide complex of Neurospora tyrosinase. (1980) FEBS Lett. 111, 232-234    Non-Patent Document 29: Luminescence of carbon monoxide hemocyanins. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2387-2389    Non-Patent Document 30: Photophysical properties of hexanuclear copper(I) and silver(I) clusters. (1992) Inorg. Chem., 31, 1941-1945    Non-Patent Document 31: Photochemical and photophysical properties of tetranuclear and hexanuclear clusters of metals with d10 and s2 electronic configurations. (1993) Acc. Chem. Res. 26, 220-226    Non-Patent Document 32: Interaction of copper(I) with nucleic acids. (1990) Int. J. Radiat. Biol. 58, 215-234    Non-Patent Document 33: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. (2002) Ang. Chem. Int. Ed. 41, 2596-2599